Petunia variety KLEPH18389

ABSTRACT

A  Petunia  plant designated KLEPH18389 is disclosed. Embodiments include seeds of  Petunia  KLEPH18389, plants of  Petunia  KLEPH18389, to plant parts of  Petunia  KLEPH18389, and methods for producing a plant by crossing  Petunia  KLEPH18389 with itself or with another variety. Embodiments also relate to  Petunia  varieties, breeding varieties, plant parts, and cells derived from  Petunia  KLEPH18389, methods for producing other  Petunia  lines or plant parts derived from  Petunia  KLEPH18389, and the  Petunia  plants, varieties, and their parts derived from use of those methods. Embodiments further include hybrid  Petunia  seeds, plants, and plant parts produced by crossing  Petunia  KLEPH18389 with another  Petunia  variety or another plant type.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application whichclaims priority to U.S. application Ser. No. 15/895,447 filed on Feb.13, 2018 which is a continuation of U.S. application Ser. No. 15/461,822filed on Mar. 17, 2017 (now U.S. Pat. No. 9,913,438), which claimspriority to U.S. Provisional Application No. 62/316,271 filed Mar. 31,2016, the contents of which all are hereby incorporated in theirentirety.

BACKGROUND

All publications cited in this application are herein incorporated byreference. Petunia is a species of flowering plants in the familySolanaceae.

Petunia can be propagated from seed, cuttings, and tissue culture. Seed,cuttings and tissue culture germination protocols for Petunia arewell-known in the art.

Petunia is an important and valuable ornamental plant. Thus, acontinuing goal of ornamental plant breeders is to develop plants withnovel characteristics, such as color, growth habit, and hardiness. Toaccomplish this goal, the breeder must select and develop plants thathave traits that result in superior Petunia varieties.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

The accompanying figures, which are herein incorporated and form a partof the specification, illustrate some, but not the only or exclusiveexample embodiments and/or features. It is intended that the embodimentsand figures disclosed herein are intended to be illustrative rather thanlimiting.

FIG. 1 shows the overall plant habit of the plant grown in a pot.

FIG. 2 shows the top view of the plant and foliage.

FIG. 3 shows a close-up of a mature flower of the plant.

FIG. 4 shows a close-up of another mature flower of the plant.

FIG. 5 shows a close-up of another mature flower of the plant.

SUMMARY

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

According to one embodiment, there is provided a Petunia plant which isvalued as breeding line enabling the development of superior ornamentalPetunia plants.

Another embodiment discloses a Petunia plant, wherein a sample ofrepresentative sample of plant tissue of said Petunia is deposited witha Budapest depository and having a spotted flower color pattern. Saidplant may also have one or more flowers that have a star pattern.

Another embodiment relates to tissue culture produced from protoplastsor cells from the Petunia plants disclosed in the subject application,wherein said cells or protoplasts are produced from a plant partselected from the group consisting of pollen, ovules, embryos,protoplasts, meristematic cells, callus, leaves, anthers, cotyledons,hypocotyl, pistils, roots, root tips, flowers, seeds, petiole, andstems.

Another embodiment relates to a plant, or a part thereof, produced byPetunia KLEPH18389, wherein the plant part comprises at least one cellof Petunia KLEPH18389.

Another embodiment relates to a tissue or cell culture of regenerablecells produced from the plant of KLEPH18389 and a Petunia plantregenerated from the tissue or cell culture of KLEPH18389.

Another embodiment relates to a method of vegetatively propagating theplant of KLEPH18389, comprising the steps of: collecting tissue or cellscapable of being propagated from a plant of KLEPH18389; cultivating saidtissue or cells of the prior step to obtain proliferated shoots orplantlets; and rooting said proliferated shoots or plantlets to obtainrooted shoots or rooted plantlets.

A further embodiment relates to a method for producing an embryo orseed, wherein the method comprises crossing a KLEPH18389 plant withanother plant and harvesting the resultant embryo or seed. For example,the other plant could either be a Petunia plant or a Calibrachoa plant.

A further embodiment relates to a method for developing a Petunia plantin a Petunia plant breeding program, comprising applying plant breedingtechniques comprising crossing, recurrent selection, mutation breeding,wherein said mutation breeding selects for a mutation that isspontaneous or artificially induced, mass selection, hybridization,open-pollination breeding, backcrossing, pedigree breeding, or geneticmarker enhanced selection to the Petunia plant of KLEPH18389, or itsparts, wherein application of said techniques results in development ofa Petunia plant.

A further embodiment relates to a method of introducing a mutation intothe genome of Petunia plant KLEPH18389, said method comprisingmutagenesis of the plant, or plant part thereof, of KLEPH18389, whereinsaid mutagenesis is selected from the group consisting of temperature,long-term seed storage, tissue culture conditions (i.e., somaclonalvariation), radiation, mutagens, and targeting induced local lesions ingenomes, and wherein the resulting plant comprises at least one genomemutation and producing plants therefrom.

A further embodiment relates to a method of editing the genome Petuniaplant KLEPH18389, wherein said method is selected from the groupcomprising zinc finger nucleases, transcription activator-like effectornucleases (TALENs), engineered homing endonucleases/meganucleases, andthe clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein9 (Cas9) system, and plants producedtherefrom.

A further embodiment relates to a Petunia seed produced by growingKLEPH18389.

A further embodiment relates to a method of producing a Petunia plant,or part thereof, by growing a seed produced on KLEPH18389.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION

Origin of KLEPH18389

KLEPH18389 originated from a cross-pollination conducted in July 2015 inLatina, Italy between the proprietary female Petunia variety‘PH-2015-1066’ (unpatented) having a darker flower color with whitespots, and the proprietary male Petunia variety ‘PH-2015-1077’(unpatented) having dark purple red with white spots flower color.

The seeds from the cross were sown and plants were grown in a greenhousefor evaluation, where an individual plant designated KLEPH18389 wasselected from the group of plants in April 2016 in Latina, Italy. In May2016, KLEPH18389 was first vegetatively propagated by terminal tipcuttings and tissue culture in Latina, Italy. KLEPH18389 was found toreproduce true to type in successive generations of asexual propagationvia tissue culture and terminal tip cuttings (vegetative cuttings) withcareful attention to uniformity of plant type and has been increasedwith continued observation for uniformity. Additionally, Petunia varietyKLEPH18389 produces viable pollen and is capable of being used as aparental line in breeding programs. KLEPH18389 exhibits a unique spottedflower color pattern and may have a star pattern on one or more flowers.

The data which define these characteristics were collected from asexualreproductions carried out in Latina, Italy. Data was collected on plantsgrown 16-weeks from cultivation from potting in a plastic greenhouse.Color references are to The R.H.S. Colour Chart of The RoyalHorticultural Society of London (R.H.S.), 5^(th) edition (2007).

TABLE 1 VARIETY DESCRIPTION INFORMATION Classification: Family:Solanaceae Botanical: Petunia hybrida Common: Petunia Designation:‘KLEPH18389’ Parentage: Female parent: The proprietary female Petuniavariety ‘PH-2015-1066’ (unpatented) Male parent: The proprietary malePetunia variety ‘PH-2012-1077’ (unpatented) Plant: Vigor: Medium/compactvigor Habit: Mounding/semi-trailing Height (from top of soil): About11.0 cm Width (horizontal plant diameter): 36.0 cm Propagation: Terminaltip cuttings and tissue culture Time to produce a finished floweringplant: About 14 weeks in the winter under short day conditions, and 7weeks in the spring Time to initiate and develop roots: 2 to 3 weeksRoot description: Moderate density, moderate branching, white rootsStems: Average number (basal): 16 Length of basal branches (from thebase of the stem to the tip): 19.0 cm Internode length: 0.5 cm to 3.0 cmDiameter of branches (from midpoint): 0.35 cm Stem color: RHS 144B(Yellow-Green) Anthocyanin: Absent Texture: Pubescent Leaves:Arrangement: Alternate Length: 3.5 cm to 4.0 cm Width: 2.0 cm to 2.5 cmShape: Obovate Apex: Acute Base: Attenuate Margin: Entire Immature leafcolor:    Upper surface: RHS N137B (Green)    Lower surface: RHS 146A(Yellow-Green) Mature leaf color:    Upper surface: RHS N137B (Green)   Lower surface: RHS 146B (Yellow-Green) Texture (both upper and lowersurfaces): Pubescent Venation pattern: Arcuate Venation color:    Uppersurface: RHS N137B (Green)    Lower surface: Between RHS 144A(Yellow-Green) and RHS    144B (Yellow-Green) Petioles:    Length: 0.5cm    Diameter: 0.2 cm    Color: RHS 144B (Yellow-Green)    Texture:Pubescent Flower buds: Shape: Irregular oblong Length: 2.5 cm to 4.0 cmDiameter: 0.6 cm Color at tight bud: RHS N187A (Greyed-Purple) and RHS151A (Yellow-Green), corresponding to the star pattern and the spots,respectively, scattered along the tube Texture: Pubescent Inflorescence:Blooming habit (flowering season): Continuously flowering during springand summer Inflorescence type: Sympodial, with monochasial growth Numberof flowers per node: 1 Fragrance: Almost absent Flowers:    Arrangement:Composed of 5 petals fused at the base    Diameter (flower face): 6.0 cm   Depth (total length of flower): 5.5 cm    Throat/Funnel:      Length: 2.0 cm       Diameter (at opening): 1.3 cm       Texture:         Inner surface: Smooth          Outer surface: Pubescent      Color:          Inner surface: RHS 203B (Black) and RHS 152D         (Yellow-Green)          Outer surface: RHS 200A (Brown) and RHS151D          (Yellow-Green)    Petals:       Color of petals, matureflower (fully opened):          Upper surface: Closest to RHS N79A(Purple) with          RHS 154D spots and star pattern color         Lower surface: RHS 79A (Purple) with RHS 150C         (Yellow-Green)          spots and star pattern color but thestar pattern color          can vary slightly from RHS 150C       Apex:Mucronate       Base: Fused       Shape: Funnelform       Margin: Entire      Strength of waviness: Weak       Degree of lobation: Weak    Calyxarrangement: Actinomorphic, composed of 5 sepals       Sepals:         Color:             Upper surface: RHS 137A (Green)            Lower surface: RHS 137C (Green)          Length: 2.0 cm         Width: 0.7 cm          Shape: Oblong          Apex: Broadlyacute          Base: Fused          Margin: Entire          Texture(both upper and lower surfaces): Pubescent Pedicels:    Color: BetweenRHS 144B (Yellow-Green) and RHS 144C    (Yellow-Green)    Length: 1.2 cmto 1.8 cm    Diameter: 0.2 cm    Texture: Pubescent Reproductive organs:Stamens:    Quantity: 5    Shape: Needle with elliptic head    Filament:      Length: 1.6 cm to 2.0 cm       Diameter: 0.01 cm       Color: RHS145C (Yellow-Green)    Anther:       Shape: Oblong       Color: RHS 145B(Yellow-Green)       Length: 0.25 cm       Diameter: 0.25 cm    Pollen:      Color: RHS N189C (Greyed-Green)       Amount: Moderate Pistils:   Quantity per flower: 1    Length: 2.2 cm    Stigma color: RHS 144A(Yellow-Green)    Style color: RHS 145B (Yellow-Green)

Disease and Pest/Insect Resistance:

No disease and pest/insect resistance observed.

Table 2 below shows a comparison between Petunia KLEPH18389 and bothparental lines.

TABLE 2 Comparison of KLEPH18389 with parental lines Female parent MaleParent Characteristic KLEPH18389 ‘PH-2015-1066’ ‘PH-2015-1077’ Flowercolor A lighter basic Darker basic flower and spot flower color color(sometime distribution and more almost black) and regular spot irregularspot distribution distribution Flower size Smaller flowers Largerflowers than the male than parent KLEPH18389

Table 3 below shows a comparison between Petunia KLEPH18389 andcommercial variety ‘KLEPH15313’, U.S. Plant Pat. No. 27,237.

TABLE 3 Comparison of KLEPH18389 with commercial variety CharacteristicKLEPH18389 ‘KLEPH15313’ Flower color Burgundy purple with yellow Violetpurple with white dots scattered on the petals; dots scattered on thehas a star pattern petals; no star pattern

Further Embodiments

Spotted Flower Color Pattern—General

The spotted flower color pattern comprises having one or more spots oneach petal. The unique spotted flower color pattern comprises spots of adifferent color than the basic color of the respective flower. The spotsmay be white, cream, yellow, yellow-green, or combinations thereof incolor. The spots may be circular or irregular in shape and may also varyin size. The spotted color pattern may be present on a base flower colorof yellow, orange, red, brown, blue, black, pink, violet, orcombinations and shades thereof and may be in combination with adifferent flower pattern comprising a star pattern or a border pattern.Thus, one embodiment includes producing a Petunia or aPetunia-Calibrachoa plant having the spotted color pattern incombination of any of the above star or border patterns, spot colors,and base flower color. A “star pattern” refers to a pattern expressed onthe flower where streaks of color longitudinally divide the pigmentedsections of the flower. The streaks are sometimes referred to as linesand may be thin or thick and solid or semi-solid. The streaks or linesradiate from the center or approximately the center of the flowertowards the outer margin of the flower. The streaks or lines may radiateall the way to the outer margin of the flower or partially towards theouter margin of the flower. The streaks or lines may vary in color andcolor patterns.

Breeding with Petunia KLEPH18389

The goal of ornamental plant breeding is to develop new, unique andsuperior ornamental plants. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selection, selfing and mutations. Therefore, a breeder will neverdevelop the same genetic variety, having the same traits from the exactsame parents.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions and further selections arethen made during and at the end of the growing season.

Breeding programs combine desirable traits from two or more varieties orvarious broad-based sources into breeding pools from which varieties aredeveloped by a variety of breeding techniques and selection of desiredphenotypes.

Using Petunia KLEPH18389 to Develop Other Plants

KLEPH18389 can also provide a source of breeding material that may beused to develop new Petunia plants and varieties. Plant breedingtechniques known in the art and used in a Petunia plant breeding programinclude, but are not limited to, recurrent selection, crossing, selfing,mass selection, bulk selection, hybridization, backcrossing, pedigreebreeding, open-pollination breeding, mutation breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, mutagenesis and transformation. Oftencombinations of these techniques are used. There are many analyticalmethods available to evaluate a new variety. The oldest and mosttraditional method of analysis is the observation of phenotypic traits,but genotypic analysis may also be used.

Additional Breeding Methods

Any plants produced using KLEPH18389 as at least one parent are also anembodiment. These methods are well-known in the art and some of the morecommonly used breeding methods are described herein. Descriptions ofbreeding methods can be found in one of several reference books (e.g.,Allard, “Principles of Plant Breeding” (1999); Vainstein, “Breeding forOrnamentals: Classical and Molecular Approaches,” Kluwer AcademicPublishers (2002); Callaway, “Breeding Ornamental Plants,” Timber Press(2000); and Bragdø, Marie, “Inter-specific Crosses in Lupinus: Cytologyand Inheritance of Flower Color,” Institute of Genetics and PlantBreeding, Agricultural College of Norway, Vollebekk, Norway (Sep. 28,1956).

Alone, or in in conjunction with various breeding techniques, othertechniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers), and the making of double haploidsmay be utilized.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which Petunia plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as pollen, ovules, embryos,protoplasts, meristematic cells, callus, leaves, anthers, cotyledons,hypocotyl, pistils, roots, root tips, seeds, flowers, petiole, shoot, orstems and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asKLEPH18389 and another different Petunia having one or more desirablecharacteristics. If the two original parents do not provide all thedesired characteristics, other sources can be included in the breedingpopulation. In the pedigree method, superior plants are selfed orintercrossed and selected in successive filial generations. In thesucceeding filial generations, the heterozygous condition gives way tohomogeneous varieties as a result of self-pollination and selection.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous variety orinbred line which is the recurrent parent. The source of the trait to betransferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent and the desirable trait transferred from the donor parent.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodcommercial characteristics and yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, aPetunia plant may be crossed with another variety to produce afirst-generation progeny plant. The first-generation progeny plant maythen be backcrossed to one of its parent varieties to create a BC₁ orBC₂. Progeny are selfed and selected so that the newly developed varietyhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the nonrecurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newPetunia varieties.

Therefore, another embodiment is a method of making a backcrossconversion of KLEPH18389, comprising the steps of crossing KLEPH18389with a donor plant comprising a desired trait, selecting an F₁ progenyplant comprising the desired trait, and backcrossing the selected F₁progeny plant to KLEPH18389. This method may further comprise the stepof obtaining a molecular marker profile of KLEPH18389 and using themolecular marker profile to select for a progeny plant with the desiredtrait and the molecular marker profile of KLEPH18389.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. KLEPH18389 is suitable for use in arecurrent selection program. The method entails individual plantscross-pollinating with each other to form progeny. The progenies aregrown and the superior progenies selected by any number of selectionmethods, which include individual plant, half-sib progeny, full-sibprogeny, and selfed progeny. The selected progenies are cross-pollinatedwith each other to form progeny for another population. This populationis planted and again superior plants are selected to cross-pollinatewith each other. Recurrent selection is a cyclical process and thereforecan be repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtainnew varieties for commercial or breeding use, including the productionof a synthetic variety. A synthetic variety is the resultant progenyformed by the intercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating plants. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Mutation Breeding

Mutation breeding is another method of introducing new traits intoPetunia KLEPH18389. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions (also known as somaclonal variation), ionizingradiation, such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137),neutrons, (product of nuclear fission by uranium 235 in an atomicreactor), Beta radiation (emitted from radioisotopes such as phosphorus32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900nm); chemical mutagens (such as base analogues (5-bromo-uracil)),related compounds (8-ethoxy caffeine), antibiotics (streptonigrin),alkylating agents (sulfur mustards, nitrogen mustards, epoxides,ethylenamines, sulfates, sulfonates such as ethyl methanesulfonate,sulfones, lactones), sodium azide, hydroxylamine, nitrous acid,methylnitrilsourea, or acridines; TILLING (targeting induced locallesions in genomes), where mutation is induced by chemical mutagens andmutagenesis is accompanied by the isolation of chromosomal DNA fromevery mutated plant line or seed and screening of the population of theseed or plants is performed at the DNA level using advanced moleculartechniques. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found inVainstein, “Breeding for Ornamentals: Classical and MolecularApproaches,” Kluwer Academic Publishers (2002); Sikora, Per, et al.,“Mutagenesis as a Tool in Plant Genetics, Functional Genomics, andBreeding” International Journal of Plant Genomics. 2011 (2011); 13pages. In addition, mutations created in other Petunia plants may beused to produce a backcross conversion of Petunia that comprises suchmutation.

Mutations that occur spontaneously can also be known asnaturally-occurring mutations. These types of mutations are furtherknown as sports, breaks, or chimeras and can be comprised of single cellmutations, branch mutations, or whole plant mutations. Any one of thesemutations can change one or more phenotypic characteristics whencompared to the original plant. Therefore, another embodiment includes anaturally-occurring genetic mutation of the plant of KLEPH18389, whereinsaid mutation is comprised from the group consisting of a single cellmutation, branch mutation, or a whole-plant mutation and plants derivedfrom said naturally-occurring mutation or mutations.

Protoplast Fusion

Also known as somatic fusion, this process can be used with KLEPH18389to create hybrids. The resulting hybrid plants have the chromosomes ofeach parent and thus the process is useful for incorporating new traits.The protoplast fusion technique is well known in the art; see forexample Hamill J. D., Cocking E. C. (1988) Somatic Hybridization ofPlants and its Use in Agriculture. In: Pais M. S. S., Mavituna F.,Novais J. M. (eds) Plant Cell Biotechnology. NATO ASI Series (Series H:Cell Biology), vol 18.

Gene Editing Using CRISPR

Targeted gene editing can be done using CRISPR/Cas9 technology (Saunders& Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as selected bacteria and archaea, to respond toand eliminate invading genetic material. Ishino, Y., et al. J.Bacteriol. 169, 5429-5433 (1987). These repeats were known as early asthe 1980s in E. coli, but Barrangou and colleagues demonstrated that S.thermophilus can acquire resistance against a bacteriophage byintegrating a fragment of a genome of an infectious virus into itsCRISPR locus. Barrangou, R., et al. Science 315, 1709-1712 (2007). Manyplants have already been modified using the CRISPR system. See forexample, U.S. Application Publication No. WO2014068346 (Gyorgy et al.,Identification of a Xanthomonas euvesicatoria resistance gene frompepper (Capsicum annuum) and method for generating plants withresistance); Martinelli, F. et al., “Proposal of a Genome Editing Systemfor Genetic Resistance to Tomato Spotted Wilt Virus” American Journal ofApplied Sciences 2014; Noman, A. et al., “CRISPR-Cas9: Tool forQualitative and Quantitative Plant Genome Editing” Frontiers in PlantScience Vol. 7 November 2016; and “Exploiting the CRISPR/Cas9 System forTargeted Genome Mutagenesis in Petunia” Science Reports Volume 6:February 2016.

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:273-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476. Therefore, it is another embodimentto use the CRISPR system on Petunia KLEPH18389 to modify traits andresistances or tolerances to pests, herbicides, diseases, and viruses.

Gene Editing Using TALENs

Transcription activator-like effector nucleases (TALENs) have beensuccessfully used to introduce targeted mutations via repair of doublestranded breaks (DSBs) either through non-homologous end joining (NHEJ),or by homology-directed repair (HDR) and homology-independent repair inthe presence of a donor template. Thus, TALENs are another mechanism fortargeted genome editing using KLEPH18389. The technique is well known inthe art; see for example Malzahn, Aimee et al. “Plant genome editingwith TALEN and CRISPR” Cell & Bioscience vol. 7 21. 24 Apr. 2017.

Therefore, it is another embodiment to use the TALENs system on Petuniavariety KLEPH18389 to modify traits and resistances or tolerances topests, herbicides, and viruses.

Other Methods of Genome Editing

In addition to CRISPR and TALENs, two other types of engineerednucleases can be used for genome editing: engineered homingendonucleases/meganucleases (EMNs), and zinc finger nucleases (ZFNs).These methods are well known in the art. See for example, Petilino,Joseph F. “Genome editing in plants via designed zinc finger nucleases”In Vitro Cell Dev Biol Plant. 51(1): pp. 1-8 (2015); and Daboussi,Fayza, et al. “Engineering Meganuclease for Precise Plant GenomeModification” in Advances in New Technology for Targeted Modification ofPlant Genomes. Springer Science+Business. pp. 21-38 (2015).

Therefore, it is another embodiment to use engineered nucleases onPetunia variety KLEPH18389 to modify traits and resistances ortolerances to pests, herbicides, and viruses.

Foreign Protein Genes: Transformation

Various promoters, targeting sequences, enhancing sequences, and otherDNA sequences can be inserted into the genome for the purpose ofaltering the expression of genes.

Many techniques for altering gene expression are well-known to one ofskill in the art, including, but not limited to, knock-outs (such as byinsertion of a transposable element such as Mu (Vicki Chandler, TheMaize Handbook, Ch. 118 (Springer-Verlag 1994)) or other geneticelements such as a FRT, Lox, or other site specific integration sites;antisense technology (see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809(1988) and U.S. Pat. Nos. 5,107,065, 5,453,566, and 5,759,829);co-suppression (e.g., Taylor, Plant Cell, 9:1245 (1997); Jorgensen,Trends Biotech., 8(12):340-344 (1990); Flavell, PNAS USA, 91:3490-3496(1994); Finnegan, et al., Bio/Technology, 12:883-888 (1994); Neuhuber,et al., Mol. Gen. Genet., 244:230-241 (1994)); RNA interference (Napoli,et al., Plant Cell, 2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp,Genes Dev., 13:139-141 (1999); Zamore, et al., Cell, 101:25-33 (2000);Montgomery, et al., PNAS USA, 95:15502-15507 (1998)), virus-induced genesilencing (Burton, et al., Plant Cell, 12:691-705 (2000); Baulcombe,Curr. Op. Plant Bio., 2:109-113 (1999)); target-RNA-specific ribozymes(Haseloff, et al., Nature, 334:585-591 (1988)); hairpin structures(Smith, et al., Nature, 407:319-320 (2000); U.S. Pat. Nos. 6,423,885,7,138,565, 6,753,139, and 7,713,715); MicroRNA (Aukerman & Sakai, PlantCell, 15:2730-2741 (2003)); ribozymes (Steinecke, et al., EMBO J.,11:1525 (1992); Perriman, et al., Antisense Res. Dev., 3:253 (1993));oligonucleotide mediated targeted modification (e.g., U.S. Pat. Nos.6,528,700 and 6,911,575); Zn-finger targeted molecules (e.g., U.S. Pat.Nos. 7,151,201, 6,453,242, 6,785,613, 7,177,766 and 7,788,044);transposable elements (e.g. Dubin, M. J., et al., Transposons: ablessing curse, Current opinion in plant biology, Vol: 42, Page: 23-29,2018 and Eric T. Johnson, Jesse B. Owens & Stefan Moisyadi (2016) Vastpotential for using the piggyBac transposon to engineer transgenicplants at specific genomic locations, Bioengineered, 7:1, 3-6) and othermethods or combinations of the above methods known to those of skill inthe art.

Additional Methods of Transformation

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the subject PetuniaKLEPH18389 plants are intended to be within the scope of the embodimentsof the application.

Introduction of a New Trait or Locus into Petunia KLEPH18389

Petunia KLEPH18389 represents a new base of genetics into which a newlocus, gene, or trait may be introgressed. Direct transformation andbackcrossing represent two important methods that can be used toaccomplish such an introgression, but additional gene editing techniquesalso exist. The term backcross conversion and single locus conversionare used interchangeably.

When the term Petunia KLEPH18389 plant is used in the context of anembodiment of the present application, this also includes any singlegene conversions of Petunia KLEPH18389. The term single gene convertedplant as used herein refers to those Petunia plants which are developedby a plant breeding technique called backcrossing wherein essentiallyall of the desired morphological and physiological characteristics of avariety are recovered in addition to the single gene transferred intothe variety.

Backcross Conversions of Petunia KLEPH18389

The term “backcrossing” as used herein refers to the repeated crossingof a hybrid progeny back to the recurrent parent, i.e., backcrossing 1,2, 3, 4, 5, 6, 7, 8, or more times to the recurrent parent. The parentalPetunia plant that contributes the gene for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental Petunia plant towhich the gene or genes from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehlman & Sleper (1994). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a Petunia plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

A backcross conversion of Petunia KLEPH18389 occurs when DNA sequencesare introduced through backcrossing (Allard, “Principles of PlantBreeding” (1999) with Petunia KLEPH18389 utilized as the recurrentparent. Both naturally occurring and transgenic DNA sequences may beintroduced through backcrossing techniques. A backcross conversion mayproduce a plant with a trait or locus conversion in at least two or morebackcrosses, including at least 2 crosses, at least 3 crosses, at least4 crosses, at least 5 crosses, and the like. Molecular marker assistedbreeding or selection may be utilized to reduce the number ofbackcrosses necessary to achieve the backcross conversion. For example,see, Openshaw, S. J., et al., Marker-assisted Selection in BackcrossBreeding, Proceedings Symposium of the Analysis of Molecular Data, CropScience Society of America, Corvallis, Oreg. (August 1994), where it isdemonstrated that a backcross conversion can be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. See, Allard, “Principles of Plant Breeding” (1999). Desiredtraits that may be transferred through backcross conversion include, butare not limited to, sterility (nuclear and cytoplasmic), fertilityrestoration, drought tolerance, nitrogen utilization, ornamentalfeatures, disease resistance (bacterial, fungal, or viral), insectresistance, and herbicide resistance. In addition, an introgression siteitself, such as an FRT site, Lox site, or other site-specificintegration site, may be inserted by backcrossing and utilized fordirect insertion of one or more genes of interest into a specific plantvariety. In some embodiments, the number of loci that may be backcrossedinto Petunia KLEPH18389 is at least 1, 2, 3, 4, or 5, and/or no morethan 6, 5, 4, 3, or 2.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes or genes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny Petunia seed byadding a step at the end of the process that comprises crossing PetuniaKLEPH18389 with the introgressed trait or locus with a different plantand harvesting the resultant first generation progeny seed.

Molecular Techniques Using Petunia KLEPH18389

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions. Traditional plant breeding has principally been the source ofnew germplasm, however, advances in molecular technologies have allowedbreeders to provide varieties with novel and much wanted commercialattributes. Molecular techniques such as transformation are popular inbreeding ornamental plants and well-known in the art. See Vainstein,“Breeding for Ornamentals: Classical and Molecular Approaches,” KluwerAcademic Publishers (2002).

Breeding with Molecular Markers

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a breeding program. The markers can also be used toselect for the genome of the recurrent parent and against the genome ofthe donor parent, in for example, a backcross breeding program. Usingthis procedure can minimize the amount of genome from the donor parentthat remains in the selected plants. It can also be used to reduce thenumber of crosses back to the recurrent parent needed in a backcrossingprogram.

The use of molecular markers in the selection process is often calledgenetic marker enhanced selection. Molecular markers may also be used toidentify and exclude certain sources of germplasm as parental varietiesor ancestors of a plant by providing a means of tracking geneticprofiles through crosses. Molecular markers, which includes markersidentified through the use of techniques such as IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs),and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing Petunia KLEPH18389. See Vainstein, “Breedingfor Ornamentals: Classical and Molecular Approaches,” Kluwer AcademicPublishers (2002).

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome. See for example, Fletcher, Richard S., et al., “QTL analysis ofroot morphology, flowering time, and yield reveals trade-offs inresponse to drought in Brassica napus” Journal of Experimental Biology.66 (1): 245-256 (2014).

QTL markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a breeding program. The use of molecular markers in theselection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of ornamental plants andPetunia KLEPH18389 and regeneration of plants therefrom is well-knownand widely published. See, for example, Valla Rego, Luciana et al., CropBreeding and Applied Technology. 1(3): 283-300 (2001); Komatsuda, T., etal., Crop Sci., 31:333-337 (1991); Stephens, P. A., et al., Theor. Appl.Genet., 82:633-635 (1991); Komatsuda, T., et al., Plant Cell, Tissue andOrgan Culture, 28:103-113 (1992); Dhir, S., et al., Plant Cell Reports,11:285-289 (1992); Pandey, P., et al., Japan J. Breed., 42:1-5 (1992);and Shetty, K., et al., Plant Science, 81:245-251 (1992). Thus, anotherembodiment is to provide cells which upon growth and differentiationproduce Petunia plants having the physiological and morphologicalcharacteristics of Petunia KLEPH18389 described in the presentapplication.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such aspollen, ovules, embryos, protoplasts, meristematic cells, callus,leaves, anthers, cotyledons, hypocotyl, pistils, roots, root tips,flowers, seeds, petiole, shoot, or stems, and the like. Means forpreparing and maintaining plant tissue culture are well-known in theart.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

One or more aspects may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the embodiments is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope. Theforegoing discussion of the embodiments has been presented for purposesof illustration and description. The foregoing is not intended to limitthe embodiments to the form or forms disclosed herein. In the foregoingDetailed Description for example, various features of the embodimentsare grouped together in one or more embodiments for the purpose ofstreamlining the disclosure. This method of disclosure is not to beinterpreted as reflecting an intention that the claimed embodimentsrequire more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description, with eachclaim standing on its own as a separate preferred embodiment.

Moreover, though the description of the embodiments has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the embodiments (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice one or more embodiments.

DEPOSIT INFORMATION

A representative sample of proprietary Petunia KLEPH18389 plant tissueof the Klemm+Sohn GmbH & Co. KG disclosed above and recited in theappended claims has been made with the Provasoli-Guillard NationalCenter for Marine Algae and Microbiota, Bigelow Laboratory for NationalSciences, 60 Bigelow Drive, East Boothbay, Me. 04544. The date ofdeposit was May 10, 2018. The NCMA No. is 201805002. The deposit ofplant tissue was taken from the same deposit maintained by Klemm+SohnGmbH & Co. KG since prior to the filing date of this application. Thedeposit will be maintained in the NCMA depository for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced ifnecessary during that period. Upon issuance, all restrictions on theavailability to the public of the deposit will be irrevocably removedconsistent with all of the requirements of 37 C.F.R. §§ 1.801-1.809.

What is claimed is:
 1. A plant of Petunia variety KLEPH18389, having aspotted flower color pattern, wherein a representative sample of planttissue of said variety was deposited under NCMA No.
 201805002. 2. Aplant, or a plant part thereof, produced by growing a plant of Petuniavariety KLEPH18389, wherein a representative sample of plant tissue ofsaid variety was deposited under NCMA No. 201805002, and wherein theplant or plant part comprises at least one cell of Petunia varietyKLEPH18389.
 3. A Petunia plant, or part thereof, having all of thephysiological and morphological characteristics of the Petunia plant ofclaim
 1. 4. A tissue or cell culture of regenerable cells produced fromthe plant or plant part of claim
 2. 5. A Petunia plant regenerated fromthe tissue or cell culture of claim 4, wherein said plant has all of themorphological and physiological characteristics of Petunia varietyKLEPH18389 listed in Table
 1. 6. A method of vegetatively propagatingthe plant of claim 1, comprising the steps of: collecting tissue orcells capable of being propagated from said plant; cultivating saidtissue or cells to obtain proliferated shoots or plantlets; and rootingsaid proliferated shoots or plantlets to obtain rooted shoots or rootedplantlets.
 7. A Petunia plant produced by growing the rooted shoots orrooted plantlets of claim
 6. 8. A method for producing a seed or anembryo, wherein the method comprises crossing Petunia varietyKLEPH18389, wherein a representative sample of plant tissue of saidvariety was deposited under NCMA No. 201805002, with a different plantand harvesting the resultant seed or embryo.
 9. A method of determiningthe genotype of the Petunia plant of claim 1, wherein said methodcomprises obtaining a sample of nucleic acids from said plant anddetecting in said nucleic acids a plurality of polymorphisms.
 10. Amethod of introducing a mutation into the genome of a KLEPH18389 plant,said method comprising mutagenesis of the plant, or plant part thereof,of claim 1, wherein said method of mutagenesis is selected from thegroup consisting of temperature, long term seed storage, somaclonalvariation, radiation, chemical agents, targeting induced local lesionsin genomes, site-directed mutagenesis, and genome editing, and whereinthe resulting plant comprises at least one genome mutation.
 11. A methodof genetically modifying the plant of claim 1, wherein said geneticmodification is selected from agrobacterium-mediated gene transfer,protoplast transformation, or biolistic transformation.
 12. A plantproduced by the method of claim 11, wherein said plant has the spottedflower color pattern.
 13. A method for producing a seed or an embryo,wherein the method comprises selfing Petunia variety KLEPH18389, whereina representative sample of plant tissue of said variety was depositedunder NCMA No. 201805002, and harvesting the resultant seed or embryo.14. A plant produced by growing the seed or embryo produced by themethod of claim 13, wherein said plant has the spotted color flowerpattern.
 15. A method for developing a Petunia plant having a spottedflower color pattern, wherein said method comprises applying plantbreeding techniques to the plant of Petunia variety KLEPH18389, having aspotted flower color pattern, wherein a representative sample of planttissue of said variety was deposited under NCMA No. 201805002, toproduce a plant having a spotted flower color pattern.
 16. The method ofclaim 15, wherein said plant breeding technique is recurrent selection.17. The method of claim 15, wherein said plant breeding technique ismass selection.
 18. The method of claim 15, wherein said plant breedingtechnique is hybridization.
 19. The method of claim 15, wherein saidplant breeding technique is open-pollination.
 20. The method of claim15, wherein said plant breeding technique is backcrossing.
 21. Themethod of claim 15, wherein said plant breeding technique is pedigreebreeding.
 22. The method of claim 15, wherein said plant breedingtechnique is mutation breeding, and wherein said mutation selected isspontaneous or artificially induced.
 23. The method of claim 15, whereinsaid plant breeding technique is marker enhanced selection.
 24. A methodof editing the genome of Petunia plant KLEPH18389, said methodcomprising editing the genome of the plant, or a part thereof, of claim1, wherein said method is selected from the group comprising zinc fingernucleases, transcription activator-like effector nucleases (TALENs),engineered homing endonucleases/meganucleases, and the clusteredregularly interspaced short palindromic repeat (CRISPR)-associatedprotein9 (Cas9) system.
 25. A Petunia plant produced by the method ofclaim
 24. 26. A Petunia seed produced by growing the plant of claim 1.27. A method of producing a Petunia plant, or part thereof, produced bygrowing the seed of claim
 26. 28. The plant of claim 1, wherein saidplant also has a star pattern on one or more flowers.